Effects of extracellular vesicles derived from oral bacteria on osteoclast differentiation and activation

doi:10.21203/rs.3.rs-1627824/v1

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Effects of extracellular vesicles derived from oral bacteria on osteoclast differentiation and activation

https://doi.org/10.21203/rs.3.rs-1627824/v1

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Dysbiosis of the oral microbiota plays an important role in the progression of periodontitis, which is characterized by chronic inflammation and alveolar bone loss, and associated with systemic diseases. Bacterial extracellular vesicles (EVs) contain various bioactive molecules and show diverse effects on host environments depending on the bacterial species. Recently, we reported that EVs derived from Filifactor alocis, a Gram-positive periodontal pathogen, had osteoclastogenic activity. In the present study, we analysed the osteoclastogenic potency and immunostimulatory activity of EVs derived from the Gram-negative periodontal pathogens Porphyromonas gingivalis and Tannerella forsythia, the oral commensal bacterium Streptococcus oralis, and the gut probiotic strain Lactobacillus reuteri. Bacterial EVs were purified by density gradient ultracentrifugation using OptiPrep^™ reagent. EVs from P. gingivalis, T. forsythia, and S. oralis increased osteoclast differentiation and osteoclstogenic cytokine expression in osteoclast precursors, whereas EVs from L. reuteri did not. EVs from P. gingivalis, T. forsythia, and S. oralis preferentially activated TLR2 rather than TLR4 or TLR9, and induced osteoclastogenesis mainly through TLR2. The osteoclastogenic effects of EVs from P. gingivalis and T. forsythia were reduced by both lipoprotein lipase and polymyxin B, an inhibitor of LPS, while the osteoclastogenic effects of EVs from S. oralis were reduced by lipoprotein lipase alone. These results demonstrate that EVs from periodontal pathogens and oral commensal have osteoclastogenic activity through TLR2 activation by lipoproteins and/or lipopolysaccharide.

Bacterial extracellular vesicle

Periodontal pathogen

Osteoclast

Toll-like receptor 2

Periodontitis is a chronic inflammatory disease initiated by polymicrobial infection of oral bacterial species, leading to tissue inflammation, alveolar bone loss, and diverse systemic diseases¹. Oral commensal bacteria have immunomodulatory activities and play an important role in host-microbe homeostasis. However, the predominance of Gram-negative periodontal pathogens such as ‘red complex’ bacteria (Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola) is known as a major cause of periodontitis². Recently, Filifactor alocis, a Gram-positive bacterium, has also been regarded as an emerging periodontal pathogen that is frequently detected in periodontal pockets in periodontitis patients and considered a co-occurrence group with periodontal pathogens, including P. gingivalis and T. forsythia³. Periodontal pathogens have various virulence factors, including microbe-associated molecular patterns (MAMPs), proteases, toxins, and complement inhibitors, that play an important role in the progression of periodontitis⁴. However, probiotic strains exhibit beneficial effects on the treatment and prevention of periodontal diseases⁵.

Osteoclasts are multinucleated cells that originate from bone marrow-derived haematopoietic precursor cells and play an important role in the regulation of bone homeostasis by resorbing bone⁶. Excessive activation of osteoclasts causes an imbalance in bone metabolism, leading to the progression of bone loss. The differentiation and activation of osteoclasts are mainly regulated by receptor activator of nuclear factor-κB ligand (RANKL), macrophage colony-stimulating factor (M-CSF), and proinflammatory cytokines such as TNF-α, IL-6 and IL-1⁷. Osteoclasts express Toll-like receptors (TLRs), which are essential pattern recognition receptors for innate immunity against microbial infections that recognize MAMPs⁸. Bacterial-derived MAMPs, such as lipopolysaccharide (LPS), lipoproteins, flagellin, DNA and RNA, can modulate osteoclast differentiation and activation through TLR signalling pathways⁹.

Bacterial extracellular vesicles (EVs) are bacteria-derived nanosized vesicles surrounded by lipid bilayers and have diverse effects on host environments. They carry various bioactive molecules including MAMPs, proteins, lipids, sugars and nucleic acids, of their originated bacteria and transfer the molecules to host cells¹⁰. The immunostimulatory activities of bacterial EVs are dependent on the profiles of their molecular components. EVs from Gram-negative bacteria have LPS, which is a representative TLR4 ligand and regarded as a major MAMP for inducing proinflammatory responses in host cells¹¹. In EVs from Gram-positive bacteria, bacterial lipoproteins are known as major TLR2 ligands¹². EVs from probiotic strains harbour immunomodulatory molecules that have beneficial properties, such as a reduction in proinflammatory cytokines and an increase in epithelial barrier functions^13,14.

Recently, we reported for the first time that EVs derived from the Gram-positive periodontal pathogen F. alocis induced osteoclast differentiation through TLR2¹⁵. In the present study, we evaluated the effects of EVs from Gram-negative periodontal pathogens P. gingivalis and T. forsythia, oral commensal bacterium Streptococcus oralis, and a gut probiotic strain Lactobacillus reuteri on osteoclast differentiation.

Isolation and characterization of bacterial EVs. EVs from periodontal pathogens (P. gingivalis, T. forsythia, and F. alocis), an oral commensal strain (S. oralis), and a gut probiotic strain (L. reuteri) were purified by buoyant density gradient ultracentrifugation. The morphology and size of the bacterial EVs were examined by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA), respectively. The TEM image showed that EVs released from P. gingivalis (Pg EVs), T. forsythia (Tf EVs), S. oralis (So EVs), L. reuteri (Lr EVs), and F. alocis (Fa EVs) were nanosized and vesicular structures without residual bacterial cells (Fig. 1A). The mean size was 199.1 nm for Pg EVs, 174.5 nm for Tf EVs, 209.5 nm for So EVs, 260.5 nm for Lr EVs, and 133.4 nm for Fa EVs (Fig. 1B). The EV protein yield (µg of EV proteins per 1 L of bacterial culture supernatant) was 393 µg for Pg EVs, 873 µg for Tf EVs, 453 µg for So EVs, 216 µg for Lr EVs, and 153 µg for Fa EVs (Fig. 1C). Next, to identify the purity of bacterial EVs, we calculated the number of EV particles/µg of EV proteins. As shown in Fig. 1D, particles/µg protein of all bacterial EVs were > 1 × 10¹⁰, which was regarded as pure EVs¹⁶. To determine the endotoxin unit (EU) of EVs from Gram-negative periodontal pathogens (Pg EVs and Tf EVs), an LAL test was conducted. As shown in Fig. 1E, endotoxins were detected both in Pg EVs and Tf EVs, and the EU/ml of the EVs was 1.5 for Pg EVs and 1.4 for Tf EVs. EVs from Gram-positive bacteria (So EVs, Lr EVs, and Fa EVs) were also examined to evaluate endotoxin contamination. No endotoxin activity was detected in EVs from Gram-positive bacteria (So EVs, Lr EVs, and Fa EVs). These results suggested that we obtained structurally intact and pure bacterial EVs.

Immunostimulatory effects of oral bacterial EVs on osteoclast precursors. Osteoclasts are multinucleated giant cells that originated from haematopoietic lineage mononuclear cells. Local infection of periodontal pathogens can cause osteoclast differentiation and bone resorption¹⁷. Recently, we determined that EVs from the Gram-positive periodontal pathogen F. alocis efficiently induce osteoclast differentiation and activation¹⁵. We used Fa EVs as a positive control to induce osteoclast differentiation in further experiments in this study. To evaluate the osteoclastogenic potencies of EVs from Gram-negative periodontal pathogens (Pg EVs and Tf EVs), an oral commensal strain (So EVs), and a gut probiotic strain (Lr EVs), committed osteoclast precursors were stimulated with bacterial EVs (10 µg/ml) without RANKL. As shown in Fig. 2A, EVs from Pg EVs and Tf EVs remarkably induced osteoclast differentiation, similar to Fa EVs. Interestingly, So EVs also induced osteoclast differentiation. However, Lr EVs did not induce differentiation to mature osteoclasts. Next, to identify the immunostimulatory effects of bacterial EVs on osteoclast precursors, we stimulated osteoclast precursors with each bacterial EV and analysed 40 cytokines in the culture supernatants using a cytokine array (Fig. 2B). Bacterial EVs, which have osteoclastogenic potencies (Pg EVs, Tf EVs, So EVs, and Fa EVs), increased the expression of G-CSF, IL-1ra, IL-6, IP-10, KC, MIP-1α, MIP-1β, MIP-2, RANTES and TNF-α in osteoclast precursors. Lr EVs increased the expression of KC, MIP-1β, and MIP-2, but did not induce major osteoclastogenic cytokines, such as TNF-α, IL-6, and IL-1β. These results indicate that EVs from oral bacteria including periodontal pathogens but not probiotic strain efficiently induce osteoclast differentiation and activation.

EVs from oral bacteria induce osteoclastogenesis preferentially via TLR2. Oral bacteria can activate TLRs in osteoclast precursors to regulate osteoclast differentiation and activation¹⁸. To determine the TLR2-, TLR4-, or TLR9-activating abilities of bacterial EVs, we used the reporter cell lines CHO/CD14/TLR2, CHO/CD14/TLR4, and HEK-Blue^™ TLR9, which highly express human TLR2, TLR4, and TLR9, respectively. We measured the TLR-activating ability of bacterial EVs at a low dose (1 µg/ml) because Pg EV-stimulated CHO/CD14 cells at 10 µg/ml were not stained with FITC-conjugated anti-CD25 antibody, which may be due to the degradation of cell surface molecules by the high proteolytic activity of Pg EVs (data not shown). As shown in Fig. 3A and B, Pg EVs, Tf EVs, So EVs, and Fa EVs highly activated TLR2. Interestingly, Pg EVs and TF EVs, EVs of Gram-negative periodontal pathogens, preferentially activated TLR2. Although TF EVs activated TLR4, the activation potential was much less than that of TLR2. Pg EVs, Tf EVs, So EVs, and Fa EVs slightly activated TLR9 (Fig. 3C). Lr EVs did not activate TLR2, TLR4 or TLR9 in our experimental conditions. To identify the role of TLR2 in bacterial EV-induced osteoclast differentiation, WT or TLR2^-/- osteoclast precursors were stimulated with bacterial EVs without RANKL. Pg EVs and Tf EVs slightly induced osteoclast differentiation in TLR2^-/- osteoclast precursors, but So EVs and Fa EVs did not induce it at all (Fig. 3D). Lr EVs did not induce osteoclast differentiation in either WT or TLR2^-/- osteoclast precursors. Likewise, ELISA data showed that major osteoclastogenic cytokines (TNF-α, IL-6 and IL-1β) were increased in WT osteoclast precursors by Pg EVs, Tf EVs, So EVs, and Fa EVs (Fig. 3E). However, in TLR2^-/- osteoclast precursors, osteoclastogenic cytokines were not induced by all EVs. These results suggest that TLR2 may be the major immune receptor for the recognition of oral bacterial EVs, leading to osteoclast differentiation and activation.

Effects of lipoproteins and LPS from oral bacterial EVs on osteoclast differentiation. Bacterial lipoproteins and LPS are known as major TLR2 and TLR4 ligands of bacterial EVs, respectively^11,12. Recently, we identified that lipoproteins of Fa EVs are major inducers of osteoclast differentiation¹⁵. To identify the role of lipoproteins and LPS in oral bacterial EVs, we incubated the EVs with lipoprotein lipase or polymyxin B and collected the EVs by ultracentrifugation to remove residual treatments. As shown in Fig. 4, both lipoprotein lipase and polymyxin B reduced the osteoclastogenic potency of Pg EVs and Tf EVs, while the osteoclastogenic potency of So EVs and Fa EVs was reduced by lipoprotein lipase alone. These results indicate that both bacterial lipoproteins and LPS of EVs from Gram-negative periodontal pathogens may act as TLR2 ligands leading to osteoclast differentiation, while lipoproteins are major TLR2 ligands of EVs from Gram-positive oral bacteria for osteoclastogenesis.

Bacteria naturally release EVs that affect host physiology without direct interaction between bacteria and host cells. EVs released from pathogens exhibit pathogenic properties, while EVs released from probiotics seem to be beneficial. In this study, we first compared the osteoclastogenic potency of EVs derived from periodontal pathogens, an oral commensal, and a probiotic strain. EVs from periodontal pathogens (Pg EVs, Tf EVs, and Fa EVs) and an oral commensal strain (So EVs) induced the expression of osteoclastogenic cytokines and osteoclast differentiation but not the probiotic strain (Lr EVs). We also demonstrated that oral bacterial EVs mainly activated TLR2 rather than TLR4 or TLR9.

Recently, we reported that EVs from Gram-positive periodontal pathogen (Fa EVs) induced osteoclast differentiation and bone resorption through TLR2¹⁵. In the present study, we observed that EVs derived from the Gram-negative periodontal pathogens P. gingivalis and T. forsythia also preferentially activate TLR2 rather than TLR4 and induce osteoclastogenesis mainly via TLR2. TLR2 signalling by P. gingivalis and T. forsythia has been reported. P. gingivalis increased TNF-α production in macrophages through TLR2 signalling, leading to alveolar bone loss¹⁹. TLR2 signalling plays an important role in T. forsythia-mediated alveolar bone loss and activation of osteoclasts²⁰. The osteoclastogenic activity of Pg EVs and Tf EVs was reduced by lipoprotein lipase treatment, suggesting that lipoproteins are active molecules, as shown in our previous study¹⁵. Jain et al. reported that P. gingivalis induced inflammation via TLR2 signalling, which was attenuated by lipoprotein lipase treatment²¹. The attenuated osteoclastogenic activity of Pg EVs and Tf EVs in TLR2^-/- osteoclast precursors supports the role of lipoproteins as TLR2 ligands. As with lipoprotein lipase treatment, the LPS inhibitor polymyxin B also reduced the osteoclastogenic activity of Pg EVs and Tf EVs, indicating that LPS was also involved. Although P. gingivalis LPS has heterogeneous lipid A structures that can activate both TLR4 and TLR2²², TLR2 may be more important than TLR4 in that P. gingivalis LPS induced alveolar bone loss through TLR2 in mice²³. Polymyxin B is known to bind to LPS, thus inhibiting the interaction of LPS with TLRs. Recently, it has been reported that EVs from Acinetobacter baumannii, a Gram-negative pathogen, directly bind to polymyxin B, leading to the protection of A. baumannii cells from lysis by polymyxin B²⁴. With regard to this finding, it can be speculated that polymyxin B can bind to LPS on EVs of P. gingivalis and T. forsythia, thus preventing the interaction of LPS with TLRs.

EVs from the oral commensal S. oralis also showed high TLR2 activation ability and osteoclastogenic potency as EVs of periodontal pathogens. Oral commensal bacteria may have negative effects on host bone metabolism. Compared with germ-free mice, specific pathogen-free mice showed increased alveolar bone loss and TRAP⁺ cells on the alveolar bone surface²⁵. Another oral commensal Streptococcus gordonii induced bone resorption in vivo, induced osteoclast differentiation and inhibited osteoblast differentiation in vitro²⁶. S. gordonii and its lipoproteins induced TNF-α and IL-1β through TLR2 in macrophages²⁷ and IL-8 in periodontal ligament cells²⁸. Oral commensal bacteria, especially viridians group streptococci (VGS), including S. oralis and S. gordonii, are commensal in human mucosa, but they are also regarded as opportunistic pathogens that can cause severe systemic diseases such as endocarditis and septic shock syndrome²⁹. Even in periodontal disease or a healthy state, oral commensal bacteria always release EVs that can affect host immunity both locally and systemically. Further studies are needed to identify the role of VGS-derived EVs in systemic diseases.

In addition to EVs derived from oral bacteria, we evaluated the effect of EVs derived from the probiotic strain L. reuteri on osteoclast differentiation. Lr EVs did not induce TLR activation, proinflammatory cytokine expression, or osteoclast differentiation. Probiotic-derived EVs have beneficial and anti-inflammatory effects in host environments¹⁴. Lr EVs inhibited LPS-induced proinflammatory responses in macrophages and splenic lymphocytes and showed protective effects against LPS-induced intestinal injury in chickens³⁰. Lactobacillus paracasei EVs reduced LPS-induced proinflammatory responses in vitro and alleviated dextran sulfate sodium (DSS)-induced colitis in mice³¹. EVs from Lactobacillus plantarum increase M2 macrophage polarization and the expression of the anti-inflammatory cytokine IL-10 in human skin organ culture³². Although Lr EVs harbour TLR2 ligands such as lipoproteins, they did not induce TLR2 activation and osteoclastogenesis in our experimental settings. The inability of L. reuteri EVs to activate TLR2 could be explained by lipoprotein properties or the presence of immunomodulatory mediators. Lipoproteins of L. reuteri may have less immunogenic structures and lyso-form lipoproteins. Lactobacillus bulgaricus produces lyso-form lipoproteins³³ that are produced by lipoprotein intramolecular transacylase (Lit)³⁴. L. reuteri has a protein (Accession: WP_086118074.1) that has sequence similarity with the Lit of L. bulgaricus (Accession: MBT8800808.1). Lr EVs harbour diverse immunomodulatory mediators, including 60-kDa chaperonin (groL), which efficiently increases anti-inflammatory cytokines IL-10 and TGF-β but does not induce the proinflammatory cytokines TNF-α and IL-12 in macrophages and dendritic cells^{30, 35}. The beneficial roles and therapeutic applications of Lr EVs on systemic bone diseases need to be further elucidated.

In summary, we analysed the osteoclastogenic activity of EVs derived from periodontal pathogens, an oral commensal bacterium, and a gut probiotic strain. EVs from the periodontal pathogens P. gingivalis and T. forsythia and the oral commensal S. oralis showed high TLR2-activating ability and osteoclastogenic potency. In contrast, EVs from the probiotic strain L. reuteri did not exhibit such properties. TLR2-activating molecules, including lipoproteins and LPS of oral bacterial EVs, may be major components necessary for inducing proinflammatory responses and osteoclast differentiation resulting in alveolar bone loss. Our findings provide a new perspective on the correlation between oral bacteria-derived EVs and alveolar bone loss in periodontitis patients.

Reagents and chemicals. Brain heart infusion (BHI), Columbia broth, Lactobacilli MRS broth, yeast extract and Bacto agar were purchased from BD Biosciences (San Jose, CA, USA). _L-cysteine, _L-arginine, resazurin, hemin, vitamin K, and N-acetylmuramic acid were purchased from Sigma (St. Louis, MO, USA). Alpha minimum essential medium (α-MEM) and phosphate-buffered saline (PBS) were purchased from Welgene (Daegu, South Korea). Penicillin/streptomycin (P/S), fetal bovine serum (FBS), and Ham’s F-12 medium were purchased from Gibco BRL (Paisley, UK). Recombinant murine soluble receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) were purchased from PeproTech (Rocky Hill, NJ, USA). G418, hygromycin B, Pam2CSK4, Escherichia coli LPS, ultrapure LPS, and ODN2006 were purchased from InvivoGen (San Diego, CA, USA). FITC anti-human CD25 mouse monoclonal antibody (clone#: M-A251) was purchased from BD Biosciences. Lipoprotein lipase from Pseudomonas species and polymyxin B were purchased from Sigma.

Bacteria and extracellular vesicles. P. gingivalis (ATCC 33277), T. forsythia (ATCC 43037) and F. alocis (ATCC 35896) were cultured at 37°C in an anaerobic chamber (10% CO₂, 10% H₂, 80% N₂). Streptococcus oralis (ATCC 9811) and Lactobacillus reuteri (ATCC 23272) were cultured at 37°C in an aerobic atmosphere. P. gingivalis was grown in BHI broth containing 5 µg/ml hemin and 1 µg/ml vitamin K. T. forsythia was grown in modified new oral spirochete broth (ATCC medium 1494 containing 0.25% yeast extract, 5 µg/ml hemin, 1 µg/ml vitamin K and 0.01 µg/ml N-acetylmuramic acid). F. alocis was grown in Columbia broth containing 5% yeast extract, 0.0025% resazurin, 5 µg/ml hemin, 1 µg/ml vitamin K, 1 µg/ml _L-cysteine and 2 µg/ml _L-arginine. S. oralis and L. reuteri were grown in BHI broth and Lactobacilli MRS broth, respectively. All bacteria were grown for 48 h. Purification and characterization of bacterial EVs were performed as previously described¹⁵. The purity and morphology of EVs were confirmed by NTA and TEM as previously described³⁶. EV protein yield was measured by a bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). The particle size and number of EVs were measured by NTA. EVs were stored at -80ºC until use.

Limulus amebocyte lysate test. Endotoxin units of bacterial EVs were measured using a Limulus amebocyte lysate (LAL) chromogenic endotoxin quantification kit (Thermo Fisher Scientific Inc.) according to the manufacturer’s instructions.

Osteoclast differentiation. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University (SNU-210602-1-2) and conducted in accordance with the guidelines and regulation of the institute. The animal experiments were conducted in accordance with the ARRIVE guidelines (https://arriveguidelines.org). C57BL6 mice (8 weeks old, male) were purchased from Orient Bio (Gyeonggi-do, Korea), and TLR2^-/- mice (C57BL/6) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). The osteoclastogenic effects of bacterial EVs were determined as previously described¹⁵. Briefly, bone marrow-derived cells were suspended in complete α-MEM (10% FBS and 1% P/S) and plated on 100-mm cell culture plates with M-CSF (30 ng/ml) for 3 days. Then, to obtain osteoclast precursors, the cells (2 × 10⁴/well) were plated on 48-well plates with M-CSF (30 ng/ml) for 1 day and cultured with RANKL (60 ng/ml) and M-CSF (30 ng/ml) for an additional 3 days. Osteoclast precursors were stimulated with the indicated stimuli in the presence of M-CSF (30 ng/ml) for 2 days. After the stimulation period, mature osteoclasts were stained for tartrate-resistant acid phosphatase (TRAP) using a TRAP staining kit (Sigma) according to the manufacturer’s instructions. TRAP⁺ cells with ≥ 3 nuclei were regarded as mature osteoclasts and counted from three independent samples. Representative images of TRAP⁺ multinucleated cells (mature osteoclasts) were obtained from three independent samples by digital inverted microscopy (DS-Ri2, Nikon, Tokyo, Japan).

Cytokine array and enzyme-linked immunosorbent assay. Osteoclast precursors were stimulated with the indicated stimuli in the presence of M-CSF (30 ng/ml) for 24 h. After the stimulation period, the culture supernatants were centrifuged (10,000 ×g, 4°C, 10 min) to exclude residual cell debris, and the supernatants were used. The cytokine expression profiles were measured using a Proteome Profiler Mouse Cytokine Array Kit, Panel A (R&D, Minneapolis, MN, USA), according to the manufacturer’s instructions. Proinflammatory cytokines (TNF-α, IL-6 and IL-1β) were measured by R&D ELISA kits.

Toll-like receptor activation assay. The TLR2- or TLR4-activating abilities of bacterial EVs were analysed using CHO/CD14/TLR2 and CHO/CD14/TLR4 cells, respectively (Dr Douglas Golenbock, Boston Medical Center, Boston, MA, USA). The cells were maintained in complete Ham’s F-12 medium containing G418 (1 mg/ml) and hygromycin B (0.4 mg/ml). CHO/CD14/TLR2 and CHO/CD14/TLR4 cells (3 ×10⁵/well) were plated in 48-well culture plates for 20 h and stimulated with the indicated stimuli for an additional 24 h. After the stimulation period, the cells were stained with FITC-conjugated anti-human CD25 antibodies. CD25⁺ cells were measured by flow cytometry (FACSCalibur^™, BD Biosciences) and analysed using FlowJo software (TreeStar, San Carlos, CA, USA). The TLR9-activating ability of bacterial EVs was analysed using HEK-Blue^™ TLR9 cells (Invitrogen) according to the manufacturer’s instructions. Briefly, the cells were incubated with the indicated stimuli for 24 h. Secreted embryonic alkaline phosphatase (SEAP) by TLR9 activation was measured by a spectrophotometer at 620 nm.

Statistical analysis. All experiments were performed at least three times. The data are represented as the mean values ± standard deviations. One-way analysis of variance (ANOVA) was performed to determine statistical significance among multiple groups with one independent variable. Two-way ANOVA performed to determine statistical significance among multiple groups with two independent variables. A p value of < 0.05 was considered statistically significant.

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Author contributions

H.Y.K., M.S., H.K., and B.C. designed the research. H.Y.K., M.S., Y.L., J.S.J., and S.A. performed all experiments and analysed and interpreted the data. H.Y.K. and B.C. wrote the main manuscript text. H.Y.K. and B.C. provided funding and supervision. All authors reviewed the manuscript.

Acknowledgements

This work was supported by grants from the National Research Foundation of Korea (NRF-2021R1A2C1003952 and NRF-2022R1C1C2007752) and the Dental Research Institute of Seoul National University.

Competing Interests

The authors have no competing financial interests to declare.

Hajishengallis, G. & Chavakis, T. Local and systemic mechanisms linking periodontal disease and inflammatory comorbidities. Nat. Rev. Immunol. 21, 426–440. https://doi.org/10.1038/s41577-020-00488-6 (2021).
Darveau, R.P. Periodontitis: a polymicrobial disruption of host homeostasis. Nat. Rev. Microbiol. 8, 481–490. https://doi.org/10.1038/nrmicro2337 (2010).
Chen, H. et al. Filifactor alocis-centered co-occurrence group associates with periodontitis across different oral habitats. Sci. Rep. 12, 9053. https://doi.org/10.1038/srep09053 (2015).
Meyle, J. & Chapple, I. Molecular aspects of the pathogenesis of periodontitis. Periodontol. 2000. 69, 7–17. https://doi.org/10.1111/prd.12104 (2015).
Nguyen, T. et al. Probiotics for periodontal health-current molecular findings. Periodontol. 2000. 87, 254–267. https://doi.org/10.1111/prd.12382 (2021).
Teitelbaum, S.L. Bone resorption by osteoclasts. Science. 289, 1504–1508. https://doi.org/10.1126/science.289.5484.1504 (2000).
Burgess, T.L. et al. The ligand for osteoprotegerin (OPGL) directly activates mature osteoclasts. J. Cell. Biol. 145, 527–538. https://doi.org/10.1083/jcb.145.3.527 (2009).
Akira, S. & Takeda, K. Toll-like receptor signalling. Nat. Rev. Immunol. 4, 499–511. https://doi.org/10.1038/nri1391 (2004).
Souza, P.P.C. & Lerner, U.H. Finding a toll on the route: The fate of osteoclast progenitors after Toll-like receptor activation. Front. Immunol. 17, 1663. https://doi.org/10.3389/fimmu.2019.01663 (2019).
Toyofuku, M. et al. Types and origins of bacterial membrane vesicles. Nat. Rev. Microbiol. 17, 13–24. https://doi.org/10.1038/s41579-018-0112-2 (2019).
Ñahui Palomino, R.A. et al. Microbiota-host communications: Bacterial extracellular vesicles as a common language. PLoS Pathog. 17, e1009508. https://doi.org/10.1371/journal.ppat.1009508 (2021).
Briaud, P. & Carroll, R.K. Extracellular vesicle biogenesis and functions in Gram-positive bacteria. Infect. Immun. 16, e00433–20. https://doi.org/10.1128/IAI.00433-20 (2020).
Dean, S.N. et al. Isolation and characterization of Lactobacillus-derived membrane vesicles. Sci. Rep 9, 877. https://doi.org/10.1038/s41598-018-37120-6 (2019).
Díaz-Garrido, N. et al. Microbiota-derived extracellular vesicles in interkingdom communication in the gut. J. Extracell. Vesicles. 10, e12161. https://doi.org/10.1002/jev2.12161 (2021).
Kim, H.Y. et al. Extracellular vesicles derived from the periodontal pathogen Filifactor alocis induce systemic bone loss through Toll-like receptor 2. J. Extracell. Vesicles. 10, e12157. https://doi.org/10.1002/jev2.12157 (2021).
Webber, J. & Clayton, A. How pure are your vesicles? J. Extracell. Vesicles. 10, https://doi.org/10.3402/jev.v2i0.19861 (2013).
Zubery, Y. et al. Bone resorption caused by three periodontal pathogens in vivo in mice is mediated in part by prostaglandin. Infect. Immun. 66, 4158–4162. https://doi.org/10.1128/IAI.66.9.4158-4162.1998 (1998).
Kajiya, M. et al. Role of periodontal pathogenic bacteria in RANKL-mediated bone destruction in periodontal disease. J. Oral. Microbiol. 8, https://doi.org/10.3402/jom.v2i0.5532 (2010).
Papadopoulos, G. et al. Macrophage-specific TLR2 signaling mediates pathogen-induced TNF-dependent inflammatory oral bone loss. J. Immunol. 190, 1148–1157. https://doi.org/10.4049/jimmunol.1202511 (2013).
Myneni, S.R. et al. TLR2 signaling and Th2 responses drive Tannerella forsythia-induced periodontal bone loss. J. Immunol. 187, 501–509. https://doi.org/10.4049/jimmunol.1100683 (2011).
Jain, S. et al. A novel class of lipoprotein lipase-sensitive molecules mediates Toll-like receptor 2 activation by Porphyromonas gingivalis. Infect. Immun. 81, 1277–1286. https://doi.org/10.1128/IAI.01036-12 (2013).
Darveau, R. P. et al. Porphyromonas gingivalis lipopolysaccharide contains multiple lipid A species that functionally interact with both Toll-like receptors 2 and 4. Infect. Immun. 72, 5041–5051. https://doi.org/10.1128/IAI.72.9.5041-5051.2004 (2004).
Kassem, A. et al. Porphyromonas gingivalis stimulates bone resorption by enhancing RANKL (Receptor Activator of NF-κB Ligand) through activation of Toll-like Receptor 2 in Osteoblasts. J. Biol. Chem. 290, 20147–20158. https://doi.org/10.1074/jbc.M115.655787 (2015).
Park, J. et al. A novel decoy strategy for polymyxin resistance in Acinetobacter baumannii. Elife. 28, e66988. https://doi.org/10.7554/eLife.66988 (2021).
Irie, K. et al. Impact of the oral commensal flora on alveolar bone homeostasis. J. Dent. Res. 2014 93, 801–806. https://doi.org/10.1177/0022034514540173 (2014).
Park, O.J. et al. Streptococcus gordonii induces bone resorption by increasing osteoclast differentiation and reducing osteoblast differentiation. Microb. Pathog. 126, 218–223. https://doi.org/10.1016/j.micpath.2018.11.005 (2019).
Kim, H.Y. et al. Lipoproteins in Streptococcus gordonii are critical in the infection and inflammatory responses. Mol. Immunol. 101, 574–584. https://doi.org/10.1016/j.molimm.2018.08.023 (2018).
Kim, A.R. et al. Streptococcus gordonii lipoproteins induce IL-8 in human periodontal ligament cells. Mol. Immunol. 91, 218–224. https://doi.org/10.1016/j.molimm.2017.09.009 (2017).
Doern, C.D. et al. It's not easy being green: the viridans group streptococci, with a focus on pediatric clinical manifestations. J. Clin. Microbiol. 48, 3829–2835. https://doi.org/10.1128/JCM.01563-10 (2010).
Hu, R. et al. Lactobacillus reuteri-derived extracellular vesicles maintain intestinal immune homeostasis against lipopolysaccharide-induced inflammatory responses in broilers. J. Anim. Sci. Biotechnol. 12, 25. https://doi.org/10.1186/s40104-020-00532-4 (2021).
Choi, J.H. et al. Lactobacillus paracasei-derived extracellular vesicles attenuate the intestinal inflammatory response by augmenting the endoplasmic reticulum stress pathway. Exp. Mol. Med. 52, 423–437. https://doi.org/10.1038/s12276-019-0359-3 (2020).
Kim, W. et al. Lactobacillus plantarum-derived extracellular vesicles induce anti-inflammatory M2 macrophage polarization in vitro. J. Extracell. Vesicles. 9, 1793514. https://doi.org/10.1080/20013078.2020.1793514 (2020).
Kurokawa, K. et al. Novel bacterial lipoprotein structures conserved in low-GC content gram-positive bacteria are recognized by Toll-like receptor 2. J. Biol. Chem. 287, 13170–13181. https://doi.org/10.1074/jbc.M111.292235 (2012).
Olatunji, S. et al. Structural basis of the membrane intramolecular transacylase reaction responsible for lyso-form lipoprotein synthesis. Nat. Commun. 12, 4254. https://doi.org/10.1038/s41467-021-24475-0 (2021).
Wang, X. et al. CD4⁺CD25⁺ Treg induction by an HSP60-derived peptide SJMHE1 from Schistosoma japonicum is TLR2 dependent. Eur. J. Immunol. 39, 3052–2065. https://doi.org/10.1002/eji.200939335 (2009).
Kim, H.Y. et al. Characterization and immunostimulatory activity of extracellular vesicles from Filifactor alocis. Mol. Oral. Microbiol. 35, 1–9. https://doi.org/10.1111/omi.12272 (2020).

No competing interests reported.

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Effects of extracellular vesicles derived from oral bacteria on osteoclast differentiation and activation

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